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Table of contents
Table of Contents
Acknowledgements
Chapter 0 General introduction
1. General introduction
2. Modeling strategy
3. Document structure
Chapter 1 Introduction to ubiquinone biosynthesis
1. General introduction to ubiquinone and its role in cellular metabolism
1. What is ubiquinone?
2. Structure of ubiquinone
3. Functions of ubiquinone
1. A lipid soluble redox agent in the electron transport chain
2. An antioxidant for membrane lipids, proteins, and DNA
3. A structural membrane lipid
2. The ubiquinone biosynthesis pathway in S. cerevisiae
1. Overview
2. Individual Coq proteins
3. Known structures of Q biosynthesis proteins
4. Coq6: Existing experimental data
1. Coq6 amino acid sequence
2. Chemical reactivity: hydroxylation and deamination
3. Protein-protein interactions
4. Clinical relevance of Coq6
3. Structures of Q biosynthesis monooxygenases
1. Introduction
2. PHBH: Holotype of Class A flavoprotein monooxygenases
1. PHBH: Global fold and FAD
2. PHBH: Catalytic cycle
3. Monooxygenases: Existing computational studies
1. Computational redesign of ligand binding based on homology models
2. Molecular dynamics studies
3. Accessible volume calculation
4. Substrate docking
5. QM/MM modeling
4. Discussion
1. Challenges of studying the Coq system and the value added of molecular modeling
1. Enzyme solubility
2. Substrate solubility
3. Enzyme redox systems
4. Enzyme interdependence
5. Conclusion: Questions addressed by the present work
Chapter 2 Computational strategy and methods
1. Introduction
2. Strategy and methods
1. From questions to techniques
2. From techniques to strategy
3. Overview of homology modeling
1. Template searching and alignment
1. The importance of finding good templates
2. Sequence search by partial pairwise methods: BLAST
3. Sequence search by complete-sequence methods: PSSMs
4. Hidden Markov model methods: Phyre2
5. Structure based searching: DALI
2. Sequence alignment
1. Pairwise alignment methods
2. Multiple sequence alignment methods
3. Progressive MSA: ClustalO
4. Iterative MSA: MAFFT-L-INS-I
3. Model building
1. MODELLER
2. I-TASSER and ROBETTA
4. Molecular dynamics
1. Molecular simulation
1. From the macroscopic to the microscopic
2. From particles of matter to systems in phase space
3. Algorithmic implementation of ensemble constraints
4. From cold crystals to warm bodies
2. Molecules: atomic structures and interatomic forces
1. Physics and functional representation: the potential energy function
2. Force-field selection: AMBER99-SB-ILDN
3. Molecular dynamics simulation code: GROMACS
4. Molecular dynamics protocols
5. Accessible volume calculation
1. Voronoi meshes: CAVER
6. Docking
1. Representing binding through docking simulations: AutoDock VINA
7. Computing resources
Chapter 3 Construction of Coq6 homology models and stability screening through molecular dynamics
1. Introduction
2. Template search
1. Sequence based search: Phyre2
2. Structure based search: DALI
3. Top templates: a structural review
1. 4K22
2. 4N9X
3. 2X3N
4. 1PBE
4. The Coq6 global fold can be divided into two regions for homology modeling: N-terminus and C-terminus
5. Coq6 contains an additional subdomain not present in known structural homologs
6. A Coq6-family MSA helps define the insert sequence
3. Model building
1. Modeling strategy: construction of a combinatorial set of multiple template models
1. Generation 1: 4K22 as the Coq6 N-terminal template; no FAD or Coq6-family insert
2. Generation 2: 2X3N as the Coq6 N-terminal template; no FAD or Coq6-family insert
2. Homology models including the insert are used to design constructs for in vivo testing
1. Generation 3: 2X3N as the Coq6 N-terminal template; with FAD and Coq6-family insert
2. Generation 3: Homology models from I-TASSER and ROBETTA
4. Molecular dynamics simulation of Generation 3 constructs
1. I-TASSER Coq6 model (2X3N based)
2. ROBETTA Coq6 model (1PBE based)
3. RATIONAL Coq6 model (2X3N, 4N9X, and 4K22 based)
4. Comparative regional RMSD summary plots
5. Conclusion
Chapter 4 Selection of Coq6 models through molecular dynamics and substrate docking
1. Introduction
2. Selection of Coq6 models by substrate docking
1. Receptor-ligand binding: induced fit vs. conformational selection
2. Receptor-ligand binding as approximated by ensemble docking
3. Preliminary study on substrate models and the Coq6 active site
4. Blind docking of 4-HP (polyprenyl length = 0)
5. Blind docking of 4-HP6 (polyprenyl length = 0)
6. Variations of tail length for computational and experimental approximations
7. Site directed docking of 4-HP with tail lengths of 1-6 isoprene units
8. Docking survey conclusion
3. Enzyme model analysis
1. Active site identification
2. Evolutionary residue conservation
3. Accessible volume calculation: CAVER
4. Molecular dynamics simulations: effective diameter of the tunnels and substrate
1. Substrate model selection
2. Tunnel diameter estimation
3. Atom selection
4. van der Waals radius corrections
5. re face tunnel 1
6. re face tunnel 2
7. si face tunnel 1
8. Conclusion: comparison of the 3 tunnel types
5. Substrate access channel characterization
1. Round 1 of docking: Channel traversability screening
1. Substrate docking into the I-TASSER Coq6 model
2. Substrate docking into the ROBETTA Coq6 model
3. Substrate docking into the RATIONAL Coq6 model
4. Conclusion of Round 1 of substrate docking
2. Round 2 of docking: the RATIONAL model and an active site geometry descriptor
6. Conclusion
Chapter 5 Testing the hypothesis of a Coq6 substrate access channel
1. Introduction
2. Review of known Coq6 mutants
1. The H. sapiens clinical mutation Coq6 G255R mutation corresponds to S. cerevisiae Coq6 G248R
3. MD and substrate docking of the G248R mutant
4. Rational design of novel mutants blocking the substrate access channel
1. MD and substrate docking of the L382E mutant
2. MD and substrate docking of the G248R-L382E double mutant
5. Experimental results
1. In vivo activity assays for Coq6 WT, G248R, L382E, and G248R-L382E
6. Conclusion
perspectives
1. Conclusion of the current work
2. Research perspectives
1. Molecular dynamics with substrate
2. Protein-protein interactions: binary pairs and protein complex architectures
3. Protein-membrane interactions
4. A phylogenetic study of the evolution of the Coq6-family insert
5. Modeling of C-terminal truncation mutants: a role in the deamination of
3-hexaprenyl-4-aminobenzoate?
6. Molecular dynamics over longer timescales
7. Substrate-enzyme assignment through systematic molecular modeling
